WO2024060463A1 - Cassette d'expression du gène du facteur viii de coagulation humaine améliorée et son utilisation - Google Patents

Cassette d'expression du gène du facteur viii de coagulation humaine améliorée et son utilisation Download PDF

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WO2024060463A1
WO2024060463A1 PCT/CN2022/143456 CN2022143456W WO2024060463A1 WO 2024060463 A1 WO2024060463 A1 WO 2024060463A1 CN 2022143456 W CN2022143456 W CN 2022143456W WO 2024060463 A1 WO2024060463 A1 WO 2024060463A1
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seq
fviii
factor viii
gene expression
aav
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Chinese (zh)
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胡晗阳
李丹丹
徐悦
陈晨
王天翼
袁龙辉
王新涛
杜增民
蒋威
吴侠
郑静
肖啸
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上海勉亦生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present disclosure relates to optimized polynucleotide sequences encoding Factor VIII (FVIII) polypeptides.
  • the present disclosure also relates to FVIII gene expression cassettes, recombinant AAV vectors and pharmaceutical compositions comprising the optimized polynucleotide sequence, and their use in the treatment of hemophilia A or acquired factor VIII deficiency.
  • Factor VIII plays a key role in the coagulation cascade by accelerating the conversion of factor X to factor Xa.
  • Hemophilia A is a congenital X-linked bleeding disorder characterized by lack of FVIII activity. Reduced FVIII activity inhibits the positive feedback loop in the coagulation cascade.
  • hemophilia A is treated by FVIII replacement therapy, ie, administration of FVIII protein (eg, intravenous infusion of plasma-derived or recombinant FVIII protein) to hemophilia A patients. Although this treatment is effective in controlling bleeding episodes, it is inherently costly due to the requirement for frequent infusions due to FVIII's short half-life (8-12 hours).
  • Adeno-associated virus (AAV)-based gene therapy is an attractive strategy to ultimately cure this disease.
  • AAV Adeno-associated virus
  • the progress of using AAV to deliver the FVIII gene is less than ideal, and the expression efficiency of FVIII in vivo is low.
  • the present disclosure provides optimized FVIII polynucleotide sequences with improved expression levels and FVIII gene expression cassette constructs with improved in vivo expression levels.
  • the inventor unexpectedly discovered that compared with the FVIII gene expression cassette with more complete elements, the FVIII gene expression cassette with partial regulatory elements (for example, enhancers, introns, especially 5'UTR) deleted was delivered via AAV vector to The present invention can be completed by maintaining a comparable or even significantly improved FVIII expression level after intracellular administration.
  • the present disclosure provides a polynucleotide encoding a Factor VIII (FVIII) polypeptide, wherein the nucleotide sequence of the polynucleotide is consistent with SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO :6 or the nucleotide sequence shown in SEQ ID NO:7 has at least 80% identity, preferably at least 85%, 90%, 95%, 99% identity.
  • FVIII Factor VIII
  • the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
  • the nucleotide sequence of the polynucleotide is shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
  • the above-described polynucleotide expresses a B domain deleted Factor VIII polypeptide (FVIII-BDD).
  • the optimized FVIII polynucleotide sequence of the present disclosure can significantly increase the expression level of FVIII protein compared to an unoptimized FVIII polynucleotide sequence.
  • the present disclosure provides a factor VIII gene expression cassette, which includes the following elements, preferably consists of the following elements: a promoter, an optional kozak sequence, a polynucleotide encoding a factor VIII polypeptide with a B domain deletion (e.g., a polynucleotide according to the first aspect), polyadenylic acid (poly A), and two ITRs located at both ends.
  • a promoter preferably consists of the following elements: a promoter, an optional kozak sequence, a polynucleotide encoding a factor VIII polypeptide with a B domain deletion (e.g., a polynucleotide according to the first aspect), polyadenylic acid (poly A), and two ITRs located at both ends.
  • the elements in the Factor VIII gene expression cassette are directly connected or indirectly connected through a linker.
  • the promoter is a ubiquitous expression promoter, a constitutive liver-specific promoter, or a synthetic liver-specific promoter.
  • the promoter is a synthetic liver-specific promoter.
  • the promoter is the liver-specific promoter shown in SEQ ID NO: 9 or SEQ ID NO: 10.
  • each of the two ITRs is independently a normal ITR or a truncated ITR.
  • the two ITRs are each independently the normal ITR shown in SEQ ID NO: 14 or the truncated ITR shown in SEQ ID NO: 15.
  • both ITRs are truncated ITRs shown in SEQ ID NO: 15.
  • the Factor VIII gene expression cassette further includes more than one of the following elements: enhancer, 5' untranslated region (5'UTR), and intron.
  • the enhancer is a constitutive enhancer or a synthetic enhancer; the enhancer shown in SEQ ID NO: 8 is preferred.
  • the intron is a constitutive intron or a synthetic intron; preferably the intron shown in SEQ ID NO: 12.
  • nucleotide sequence of the Factor VIII gene expression cassette is as shown in SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.
  • the present disclosure provides a recombinant AAV vector, which includes: the Factor VIII gene expression cassette according to the second aspect and an AAV capsid protein.
  • the AAV capsid protein is a natural AAV capsid protein or an artificially modified AAV capsid protein; preferably, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11.
  • the present disclosure provides use of the recombinant AAV vector according to the third aspect in the preparation of a medicament for preventing or treating hemophilia A or acquired factor VIII deficiency.
  • the present disclosure provides a medicament comprising the recombinant AAV vector according to the third aspect and optional excipients.
  • excipients include salts, organics, and surfactants.
  • the drug is administered by systemic or local routes, such as oral, rectal, transmucosal, intranasal, inhalation, intrabuccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intradermal, intrapleural, intracerebral, and intraarticular), intralymphatic, topical, intralesional administration.
  • systemic or local routes such as oral, rectal, transmucosal, intranasal, inhalation, intrabuccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intradermal, intrapleural, intracerebral, and intraarticular), intralymphatic, topical, intralesional
  • the present disclosure provides a method for preventing or treating hemophilia A or acquired factor VIII deficiency, comprising administering to a subject in need thereof a therapeutically effective amount of a polynucleotide according to the first aspect , the Factor VIII gene expression cassette according to the second aspect, the recombinant AAV vector according to the third aspect, the medicine according to the fifth aspect, and/or the host cell according to the eighth aspect.
  • the present disclosure provides a method for delivering Factor VIII gene into target cells, including: 1) packaging the Factor VIII gene expression cassette according to the second aspect in an AAV capsid protein to form a method according to the second aspect.
  • the target cells are ex vivo cells. In one embodiment, the target cells are cells in vivo.
  • the present disclosure provides a host cell infected with the recombinant AAV vector according to the third aspect.
  • the present disclosure provides a method for producing a recombinant AAV vector, comprising introducing a polynucleotide according to the first aspect into a mammalian host cell, the mammalian host cell comprising an AAV rep gene, an AAV cap gene and viral replication accessory genes.
  • Figure 1 shows the components of the gene expression cassette before and after codon optimization of the FVIII gene: inverted terminal repeat (ITR) sequence (SEQ ID NO: 14), enhancer (SEQ ID NO: 8), and liver-specific promoter LXP2.
  • ITR inverted terminal repeat
  • SEQ ID NO: 14 enhancer
  • SEQ ID NO: 8 liver-specific promoter LXP2.
  • SEQ ID NO: 9 liver-specific promoter LXP3.3
  • SEQ ID NO: 10 or liver-specific promoter LXP3.3
  • SEQ ID NO: 10 or liver-specific promoter LXP3.3 (SEQ ID NO: 10), 5'UTR (SEQ ID NO: 11), intron (SEQ ID NO: 12), Kozak sequence ( GCCACC), human FVIII-encoding cDNA (FVIII-BDD, SEQ ID NO: 3) or codon-optimized human FVIII-encoding cDNA (FVIII-BDD-Opti, SEQ ID NO: 4-7), poly(A) A).
  • Figure 2 shows the expression level of FVIII protein after transfection of Huh7 cells with the FVIII gene expression cassette plasmid having the components shown in Figure 1. Shown are the coagulation activity test results of the Huh7 cell culture supernatant transfected with the gene expression cassette.
  • Figure 3 shows the FVIII gene expression cassette composed of different combinations of codon-optimized FVIII gene (SEQ ID NO: 7) and expression regulatory elements.
  • Figure 4 shows the FVIII protein expression level after transfection of Huh7 cells with the FVIII gene expression cassette plasmid shown in Figure 3. Shown are the coagulation activity test results of the Huh7 cell culture supernatant transfected with the gene expression cassette.
  • Figure 5A shows the virus yield of adeno-associated virus (AAV) packaging of each FVIII gene expression cassette shown in Figure 3 under the same production conditions.
  • AAV adeno-associated virus
  • Figure 5B shows the results of alkaline gel electrophoresis of samples produced by DNA extraction of adeno-associated virus (AAV) packaging each FVIII gene expression cassette shown in Figure 3.
  • AAV adeno-associated virus
  • FIG. 6 shows the FVIII protein expression level after infection of Huh7 cells with adeno-associated virus (AAV) packaging each FVIII gene expression cassette shown in Figure 3. Shown are the coagulation activity test results of Huh7 cell culture supernatants infected with AAV carrying gene expression cassettes.
  • AAV adeno-associated virus
  • Figure 7 shows long-term FVIII activity (as a percentage of normal human FVIII activity) in 5 FVIII knockout mice after intravenous injection of AAV32.1/LXP2.1-F8X1Co4.3.
  • nucleic acid or polynucleotide sequences listed herein are in single-stranded form and are oriented from 5' to 3', left to right.
  • the nucleotides and amino acids provided in this article adopt the format recommended by the IUPACIUB Biochemical Nomenclature Committee, and use single-letter codes or three-letter codes for amino acids.
  • the terms "patient” and “subject” are used interchangeably and in their conventional sense and include any animal suffering from or susceptible to a hemorrhagic disorder or bleeding condition that requires and/or desires control of bleeding, It can be treated, ameliorated or prevented by administering FVIII to a subject (eg, hemophilia A and acquired FVIII deficiency (eg, due to autoantibodies to FVIII or hematological malignancies)).
  • a subject eg, hemophilia A and acquired FVIII deficiency (eg, due to autoantibodies to FVIII or hematological malignancies)).
  • Such subjects are typically mammals (e.g., laboratory animals such as rats, mice, guinea pigs, rabbits, primates, etc.), farm or commercial animals (e.g., cows, horses, goats, donkeys, sheep etc.) or domestic animals (such as cats, dogs, ferrets, etc.).
  • the subject is a primate subject, a non-human primate subject (eg, a chimpanzee, baboon, monkey, gorilla, etc.), or a human.
  • a subject of the present disclosure may be a subject known or believed to have a bleeding disorder or risk of a bleeding condition in need and/or desired control.
  • subjects according to the present disclosure may also include subjects previously unknown or suspected to have a bleeding disorder or risk of a bleeding condition that requires or is desirable to control.
  • the subject may be a laboratory animal and/or an animal model of the disease.
  • Subjects include males and/or females of any age, including newborns, infants, adults and elderly subjects.
  • subjects can be infants (e.g., less than about 12 months, 10 months, 9 months, 8 months, 7 months, 6 months or less age), toddlers (e.g., at least about 12, 18 or 24 months and/or less than about 36, 30 or 24 months) or children (e.g., at least about 1, 2, 3, 4 or 5 years old and/or less than about 14 years old, 12, 10, 8, 7, 6, 5 or 4 years old).
  • the subject is a human subject of about 0 to 3, 4, 5, 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 3 to 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 6 to 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 9 to 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 12 to 18, 24, 36, 48 or 60 months of age, a human subject of about 18 to 24, 30, 36, 48 or 60 months of age, or a human subject of about 24 to 30, 36, 48 or 60 months of age.
  • treatment includes: (1) inhibiting a condition, disease or disorder, that is, preventing, reducing or delaying the progression of the disease or its recurrence or the development of at least one clinical or subclinical symptom thereof; or (2) Alleviating a disease, that is, causing resolution of a condition, disease or condition or at least one of its clinical or subclinical symptoms.
  • a therapeutically effective amount is an amount that provides some improvement or benefit to a subject.
  • a therapeutically effective amount of a drug suitable for treating hemophilia A or acquired factor VIII deficiency may be an amount capable of preventing or ameliorating one or more symptoms associated with hemophilia A or acquired factor VIII deficiency. .
  • the term "improvement” refers to an improvement in a symptom associated with a disease, and may refer to an improvement in at least one parameter that measures or quantifies the symptom.
  • preventing a condition, disease or disorder includes preventing, delaying or reducing the incidence and/or likelihood of the occurrence of at least one clinical or subclinical symptom of the condition, disease or disorder developing in a subject,
  • the subject may have or be susceptible to the condition, disease, or disorder but has not yet experienced or exhibited clinical or subclinical symptoms of the condition, disease, or disorder.
  • preventing hemophilia A means reducing the number and/or severity of bleeding episodes compared to the number and/or severity of bleeding episodes that would occur in the absence of preventive treatment.
  • topical administration or “topical route” refers to administration having a local effect.
  • a measurable value such as an amount of a polypeptide, dosage, time, temperature, enzyme activity or other biological activity, etc.
  • the term "about” as used herein is intended to encompass variations of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5% or even ⁇ 0.1% of the specified amount.
  • a polynucleotide or polypeptide sequence of the present disclosure means that it consists of the sequence (e.g., SEQ ID NO) and the 5' and/or 3' or N terminus and/or of the sequence. or a polynucleotide or polypeptide consisting of a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) additional nucleotides or amino acids on the C-terminus, wherein The additional nucleotides or amino acids do not result in a substantial change in the function of the polynucleotide or polypeptide.
  • substantially altered means an increase or decrease in the ability to express the encoded polypeptide by at least about 50% or more as compared to the expression level of the polynucleotide consisting of the sequence.
  • substantially altered as applied to a polypeptide of the present disclosure refers to an increase or decrease in coagulation-stimulating activity of at least about 50% or more as compared to the activity of the polypeptide consisting of the recited sequence.
  • the terms “enhance,” “increase,” or “improve” refer to an increase in a specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold or even 15-fold.
  • the terms “inhibit,” “reduce,” or “reduce” refer to a decrease or decrease in a specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80% , 90%, 95% or more. In one embodiment, “inhibit,” “reduce,” or “reduce” results in little or essentially undetectable activity (eg, less than about 10% or even 5%).
  • the efficacy of treating hemophilia A or acquired Factor VIII deficiency by the methods of the present disclosure can be determined by detecting clinical improvement indicated by changes in symptoms and/or clinical parameters of the subject.
  • operably linked refers to a relationship between a first nucleotide sequence (e.g., a gene) and a second nucleotide sequence (e.g., a regulatory control element) that allows the second nucleotide sequence
  • the sequence affects one or more properties (eg, transcription rate) associated with the first nucleotide sequence.
  • a promoter is operably linked to a coding sequence, such as directly (no additional nucleotides are included between the promoter and the coding sequence) or indirectly via a linker (eg, a nonfunctional linker).
  • nucleic acid As used herein, “nucleic acid,” “nucleotide sequence” and “polynucleotide” are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (eg, chemically synthesized) DNA or RNA and chimeras of RNA and DNA.
  • the terms “nucleic acid,” “nucleotide sequence,” and “polynucleotide” refer to a chain of nucleotides without regard to chain length. Nucleic acids can be double-stranded or single-stranded. When single-stranded, the nucleic acid can be either the sense strand or the antisense strand.
  • Nucleic acids can be synthesized using oligonucleotide analogs or derivatives (eg, inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids with altered base pairing abilities or increased nuclease resistance.
  • the present disclosure also provides a nucleic acid that is the complement (which may be a complete complement or a partial complement) of a nucleic acid, nucleotide sequence or polynucleotide of the present disclosure.
  • isolated polynucleotide is a nucleotide sequence (eg, DNA or RNA) that is not directly linked to a nucleotide sequence (one at the 5' terminus and the other in the naturally occurring genome of the organism from which it is derived). one at the 3' end).
  • the isolated nucleic acid includes some or all of the 5' non-coding (eg, promoter) sequences immediately adjacent to the coding sequence.
  • the term includes recombinant DNA incorporated, for example, into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryotic or eukaryotic organism, or as a separate molecule (e.g., by PCR or restriction enzymes).
  • cDNA or genomic DNA fragments produced by nuclease treatment exist independently of other recombinant DNA sequences. It also includes recombinant DNA that is part of a hybrid nucleic acid encoding an additional polypeptide or peptide sequence.
  • An isolated polynucleotide that includes a gene is not a segment of the chromosome that includes such a gene, but rather includes the coding and regulatory regions associated with the gene, but without the additional genes naturally occurring on the chromosome.
  • fragment applied to polynucleotides will be understood to refer to a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence, and comprising (substantially consisting of and/or consisting of) a nucleotide sequence of consecutive nucleotides that are consistent or almost consistent (e.g., 90%, 92%, 95%, 98%, 99% consistent) with the reference nucleic acid or nucleotide sequence.
  • nucleic acid fragments according to the present disclosure may be included in larger polynucleotides of which they are a component, where appropriate.
  • such a fragment may comprise (substantially consisting of and/or consisting of) an oligonucleotide having a length of at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or more consecutive nucleotides of a nucleic acid or nucleotide sequence according to the present disclosure.
  • isolated may refer to nucleic acids, nucleic acids, nucleic acids, nucleic acids, nucleic acids, nucleic acids, nucleic acids, nucleic acids, and/or nucleic acids that are substantially free of cellular material, viral material, and/or culture media (when produced by recombinant DNA technology), or chemical precursors or other chemicals (when chemically synthesized). nucleotide sequence or polypeptide.
  • an "isolated fragment” is a fragment of a nucleic acid, nucleotide sequence, or polypeptide that is not a naturally occurring fragment and would not be found in its natural state. "Isolated” does not mean that the preparation is technically pure (homogeneous), but that it is sufficiently pure to provide the polypeptide or nucleic acid in a form useful for the intended purpose.
  • fragment as applied to a polypeptide will be understood to mean an amino acid sequence that is reduced in length relative to a reference polypeptide or amino acid sequence and contains (consists essentially of and/or consists of) the same sequence of amino acids as said reference polypeptide or amino acid sequence.
  • Amino acid sequences of contiguous amino acids whose sequences are identical or nearly identical (eg, 90%, 92%, 95%, 98%, 99% identical).
  • Such polypeptide fragments according to the present disclosure may, where appropriate, be included within the larger polypeptide of which they are a constituent. In some embodiments, such fragments may comprise (consist essentially of and/or consist of) at least about 4, 6, 8, 10, 12, 15, Peptides of 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or more consecutive amino acid lengths.
  • a “vector” is any nucleic acid molecule used for cloning and/or transferring nucleic acids into cells.
  • a vector can be a replicon to which another nucleotide sequence can be attached to allow replication of the attached nucleotide sequence.
  • a “replicon” may be any genetic element (eg, plasmid, phage, cosmid, chromosome, viral genome) that is an autonomous unit of nucleic acid replication in vivo (ie, capable of replicating under its own control).
  • the term “vector” includes viral and non-viral (eg, plasmid) nucleic acid molecules that introduce nucleic acids into cells in vitro, ex vivo, and/or in vivo.
  • a wide variety of vectors known in the art can be used to manipulate nucleic acids, incorporate response elements and promoters into genes, and the like. For example, insertion of nucleic acid fragments corresponding to the response element and promoter into a suitable vector can be achieved by ligating the suitable nucleic acid fragment into a selected vector with complementary binding termini. Alternatively, the ends of the nucleic acid molecules can be enzymatically modified, or any site can be created by joining nucleotide sequences (linkers) to the nucleic acid ends.
  • Such vectors can be engineered to contain sequences encoding a selectable marker, which facilitates selection of cells containing the vector and/or cells that have incorporated the nucleic acid of the vector into the cellular genome.
  • a "recombinant" vector refers to a viral or non-viral vector that contains one or more heterologous nucleotide sequences (i.e., a transgene), e.g., two, three, four, five or more heterologous nucleotides sequence.
  • Viral vectors have been used in a variety of gene delivery applications in cells as well as in living animal subjects.
  • Viral vectors that may be used include, but are not limited to, retroviral, lentiviral, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpesvirus, Epstein-Barr virus, and adenovirus vectors.
  • Non-viral vectors include plasmids, liposomes, charged lipids (cytofectins), nucleic acid-protein complexes and biopolymers.
  • the vector may also contain one or more regulatory regions and/or selectable markers that can be used to select, measure and monitor the outcome of nucleic acid transfer (delivery to specific tissues, duration of expression, etc.).
  • the vector can be introduced into the desired cells by methods known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome) fusion), using gene guns or nucleic acid vector transporters (see, e.g., Wu et al., J. Biol. Chem. 267:963 (1992); Wu et al., J. Biol. Chem. 263:14621 (1988); and Hartmut et al. Canadian Patent Application No. 2,012,311 filed March 15, 1990).
  • methods known in the art such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome) fusion
  • gene guns or nucleic acid vector transporters see, e.g., Wu et al., J. Biol. Chem. 267:963 (1992); Wu et al., J
  • nucleic acids in vivo
  • cationic oligopeptides e.g., WO95/21931
  • peptides derived from nucleic acid binding proteins e.g., WO96/25508
  • cationic polymers e.g., WO96/25508
  • Vectors that are naked nucleic acids can also be introduced in vivo (see U.S. Patent Nos. 5,693,622, 5,589,466, and 5,580,859).
  • Receptor-mediated nucleic acid delivery methods can also be used (Curiel et al., Hum. Gene Ther. 3:147 (1992); Wu et al., J. Biol. Chem. 262:4429 (1987)).
  • protein and “polypeptide” as used herein are used interchangeably and include both peptides and proteins.
  • a "fusion protein” is when two heterologous nucleotide sequences encoding two (or more) different polypeptides, or fragments thereof, not found fused together in nature, are fused together in the correct translation reading frame , the resulting peptides.
  • Illustrative fusion polypeptides include polypeptides of the present disclosure (or fragments thereof) and glutathione-S-transferase, maltose-binding protein, or reporter proteins (e.g., green fluorescent protein, beta-glucuronidase, beta-galactopyranoside enzyme, luciferase, etc.), hemagglutinin, c-myc, FLAG epitope, etc., or all or part of the fusion.
  • a “functional” polypeptide or “functional fragment” is a substance that substantially retains at least one biological activity normally associated with the polypeptide (e.g., angiogenic activity, protein binding, ligand or receptor binding). In one embodiment, a “functional" polypeptide or “functional fragment” substantially retains all activities possessed by an unmodified peptide. By “substantially retaining" biological activity, it is meant that the polypeptide retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 99% or more of the biological activity of the native polypeptide (and may even have a higher level of activity than the native polypeptide).
  • non-functional polypeptide is a polypeptide that exhibits little or substantially no detectable biological activity normally associated with a polypeptide (e.g., at most an insignificant amount, such as less than about 10% or even 5%). Biological activities such as protein binding and angiogenic activity can be measured using assays well known in the art and as described herein.
  • expressing means that the sequence is transcribed and optionally translated. Generally, in accordance with the present disclosure, expression of a coding sequence of the disclosure will result in the production of a polypeptide of the disclosure. Whole-body expressed polypeptides or fragments can also function in intact cells without the need for purification.
  • AAV adeno-associated virus
  • AAV type 1 includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV 8 type, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV and any other AAV now known or later discovered.
  • BERNARD N. FIELDS et al. VIROLOGY, Volume 2, Chapter 69 (4th ed., Lippincott-Raven Publishers).
  • a number of additional AAV serotypes and clades have been identified (see, eg, Gao et al., (2004) J. Virol. 78:6381-6388), which are also encompassed by the term "AAV”.
  • genomic sequences of various AAVs and autonomous parvoviruses as well as the sequences of ITRs, Rep proteins and capsid subunits are known in the art. These sequences can be found in the literature or in public databases, such as GenBank, see, for example, GenBank Accession Nos.
  • a “recombinant AAV vector genome” or “rAAV genome” is an AAV genome (i.e., vDNA) comprising at least one inverted terminal repeat (e.g., one, two, or three inverted terminal repeat sequences) and one or more heterologous nucleotide sequences.
  • rAAV vectors typically retain the 145 base terminal repeats (TRs) in cis to produce the virus; however, modified AAV TRs and non-AAV TRs (including partially or completely synthetic sequences) can also be used for this purpose. All other viral sequences are unnecessary and can be provided in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158: 97).
  • rAAV vectors optionally contain two TRs (e.g., AAV TRs), which will typically be at the 5’ and 3’ ends of the heterologous nucleotide sequence, but not necessarily adjacent to it. TRs can be identical to or different from each other.
  • the vector genome may also contain a single ITR at its 3’ or 5’ end.
  • terminal repeat includes any viral terminal repeat or synthesis that forms a hairpin structure and serves as an inverted terminal repeat (i.e., mediates desired functions such as replication, viral packaging, integration and/or proviral rescue, etc.) sequence.
  • TR can be AAV TR or non-AAV TR.
  • non-AAV TR sequences such as those of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or sequences of the SV40 hairpin that serve as the origin of SV40 replication, Can be used as TRs, they can be further modified by truncation, substitution, deletion, insertion and/or addition.
  • TR may be partially or fully synthetic, such as the "double D sequence" described in Samulski et al., US Pat. No. 5,478,745.
  • AAV terminal repeat or “AAV TR” may be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or those now known or hereafter discovered Any other AAV (see e.g. Table 1).
  • AAV terminal repeats need not have native terminal repeat sequences (e.g., native AAV TR sequences can be altered by insertions, deletions, truncations, and/or missense mutations) as long as the terminal repeats mediate the desired function such as replication, viral packaging , integration and/or original virus rescue, etc.
  • rAAV particle and "rAAV viral particle” are used interchangeably herein.
  • rAAV particles or "rAAV viral particles” comprise the rAAV vector genome packaged within an AAV capsid.
  • the AAV capsid structure is described in more detail in BERNARD N. FIELDS et al., VIROLOGY, Volume 2, Chapters 69 & 70 (4th edition, Lippincott-Raven Publishers).
  • biological activity or “activity” is determined by reference to a standard, e.g., from human plasma.
  • a standard e.g., from human plasma.
  • the standard may be (CSL Behring). The biological activity of this standard was taken as 100%.
  • Factor VIII protein or “FVIII protein” includes wild-type FVIII proteins as well as naturally occurring or man-made proteins (eg, B domain deleted proteins).
  • the FVIII proteins of the present disclosure may further include mutant forms of FVIII known in the literature.
  • the FVIII proteins of the present disclosure also include any other naturally occurring human FVIII proteins or artificial human FVIII proteins now known or later identified, as well as their derivatives and active fragments/active domains known in the art.
  • FVIII amino acid sequences of FVIII from various mammalian species are available from sequence databases such as GenBank. Examples of FVIII sequences are shown in the table below.
  • the FVIII proteins of the present disclosure also include pharmacologically active forms of FVIII, which are molecules from which the signal peptide has been removed and the B domain has been deleted by the action of a protease (or engineered from the protein by removing it at the nucleic acid level ionized out), producing two discontinuous polypeptide chains (light chain and heavy chain) of FVIII that fold into functional FVIII coagulation factor.
  • pharmacologically active forms of FVIII are molecules from which the signal peptide has been removed and the B domain has been deleted by the action of a protease (or engineered from the protein by removing it at the nucleic acid level ionized out), producing two discontinuous polypeptide chains (light chain and heavy chain) of FVIII that fold into functional FVIII coagulation factor.
  • B domain deleted forms of human FVIII are known, including the frequently used SQ version in which the residues between S743 and Q1638 are deleted.
  • the amino acid sequence of the human FVIII protein is well known in the art and can be found in GenBank accession number AAA52484.
  • the human FVIII protein is 2351 amino acids in length and consists of a signal peptide (residues 1-19), a heavy chain (residues 20-759), a B domain (residues 760-1332), and a light chain (residues 1668-1668). 2351) composition.
  • the amino acid sequence of the human FVIII protein without a signal peptide is disclosed below (SEQ ID NO: 1).
  • half-life is a broad term that includes the usual and customary meaning as well as the usual and customary meaning found in the FVIII scientific literature. Specifically included in this definition are measurements of parameters related to FVIII, which define the post-infusion time taken to reduce from the initial value measured at the time of infusion to half of the initial value.
  • the half-life of FVIII can be measured in blood and/or blood components in various immunoassays using antibodies to FVIII, as is well known in the art and described herein.
  • half-life can be measured as a decrease in FVIII activity using functional assays including standard coagulation assays, as is well known in the art and as described herein.
  • a "transformed" cell is a cell that has been transformed, transduced and/or transfected with a nucleic acid molecule encoding the FVIII protein of the present disclosure, including but not limited to FVIII protein vectors constructed using recombinant DNA technology.
  • bleeding disorder reflects any defect of cellular, physiological or molecular origin, congenital, acquired or induced, in which bleeding occurs.
  • coagulation factor deficiencies e.g., hemophilia A and B or deficiencies of coagulation factors XI, VII, VIII or IX
  • coagulation factor inhibitors e.g., platelet insufficiency, thrombocytopenia, von Willebrand's disease (von Willebrand's disease) or bleeding caused by surgery or trauma.
  • Excessive bleeding also occurs in subjects with a normal functioning blood coagulation cascade (without coagulation factor deficiencies or inhibitors to any coagulation factor) and may be caused by platelet insufficiency, thrombocytopenia, or von Willebrand syndrome disease caused.
  • the bleeding may be similar to that caused by hemophilia because the hemostatic system (as in hemophilia) lacks or has an abnormality in the necessary clotting "compounds" (such as platelets or von Willebrand factor protein) , causing major bleeding.
  • normal hemostatic mechanisms may be overwhelmed by the need for immediate hemostasis, and bleeding may occur despite normal hemostatic mechanisms.
  • Radical retropubic prostatectomy is a routine surgery for subjects with localized prostate cancer. Surgery is often complicated by significant and sometimes massive blood loss. Considerable blood loss during prostatectomy is primarily related to the complex anatomy, which has various densely vascularized sites where surgical hemostasis is not readily available, and may result in large areas of diffuse bleeding. Furthermore, intracerebral hemorrhage is the least treatable form of stroke and is associated with high mortality and hematoma growth in the first hours after intracerebral hemorrhage. Another situation that may cause problems in the setting of poor hemostasis is when subjects with normal hemostatic mechanisms are treated with anticoagulation to prevent thromboembolic disease. Such treatments may include heparin, other forms of proteoglycans, warfarin, or other forms of vitamin K antagonists, as well as aspirin and other platelet aggregation inhibitors.
  • the bleeding is associated with hemophilia. In another embodiment, the bleeding is associated with acquired inhibitor hemophilia. In another embodiment, the bleeding is associated with thrombocytopenia. In another embodiment, the bleeding is associated with von Willebrand’s disease. In another embodiment, the bleeding is associated with severe tissue damage. In another embodiment, the bleeding is associated with severe trauma. In another embodiment, the bleeding is related to surgery. In another embodiment, the bleeding is associated with laparoscopic surgery. In another embodiment, the bleeding is associated with hemorrhagic gastritis. In another embodiment, the bleeding is massive uterine bleeding. In another embodiment, the bleeding occurs in an organ with limited possibilities for mechanical hemostasis. In another embodiment, the bleeding occurs in the brain, inner ear area, or eye. In another embodiment, the bleeding is associated with the procedure of taking a biopsy. In another embodiment, the bleeding is associated with anticoagulant therapy.
  • the present disclosure relates to a codon-optimized polynucleotide sequence encoding FVIII-BDD (nucleic acid optimized FVIII-BDD-Opti) for expression in humans.
  • the B domain-deleted FVIII (FVIII-BDD) protein is a modified FVIII protein with increased glycosylation (International Patent WO2016127057; amino acid sequence SEQ ID NO: 2 and nucleotide sequence SEQ ID NO: 3).
  • a codon-optimized sequence includes, consists essentially of, or consists of a sequence that is at least 90% identical to one of SEQ ID NO: 4 and/or SEQ ID NO: 6, e.g., to SEQ ID NO: 4
  • the nucleotide sequences of NO:4 and/or SEQ ID NO:6 are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sex.
  • the polynucleotide sequence optimizer has 95%-100% sequence identity with any one of SEQ ID NO:4 and/or SEQ ID NO:6.
  • the nucleic acid optimized to encode FVIII-BDD has a reduced CpG content compared to the wild-type nucleic acid encoding FVIII-BDD (SEQ ID NO: 3). In certain embodiments, the nucleic acid optimized has at least 20 fewer CpGs than the wild-type nucleic acid encoding FVIII-BDD.
  • the nucleic acid optimized has no more than 10 CpGs, no more than 9 CpGs, no more than 8 CpGs, no more than 7 CpGs, no more than 6 CpGs, no more than 5 CpGs, no more than 4 CpGs, no more than 3 CpGs, no more than 2 CpGs, or no more than 1 CpG. In certain embodiments, the nucleic acid optimized has at most 4 CpGs, 3 CpGs, 2 CpGs, or 1 CpG. In certain embodiments, the nucleic acid optimized has no CpGs.
  • an optimized nucleic acid encoding FVIII-BDD has reduced CpG content compared to a wild-type nucleic acid encoding FVIII-BDD, and such reduced CpG nucleic acid variant is consistent with SEQ ID NO: 5 and/or or any one of SEQ ID NO:7 having 90% or greater sequence identity.
  • the CpG-reduced nucleic acid variant is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or higher sequence identity.
  • the CpG-reduced nucleic acid optimizer has 90%-95% sequence identity with any one of SEQ ID NO:5 and/or SEQ ID NO:7.
  • the CpG-reduced nucleic acid optimizer has 95%-100% sequence identity with any one of SEQ ID NO:5 and/or SEQ ID NO:7.
  • an optimized nucleic acid encoding a reduced CpG of FVIII-BDD is set forth in any one of SEQ ID NO: 5 and/or SEQ ID NO: 7.
  • a nucleic acid variant encoding a FVIII-BDD protein is at least 75% identical to a B domain deleted wild-type human FVIII nucleic acid. In certain embodiments, a nucleic acid variant encoding a FVIII-BDD protein is about 75-95% identical to a B domain deleted wild-type human FVIII nucleic acid, e.g., about 75%, 76%, 77%, 78%, 79 %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% same .
  • the nucleic acids and variants encoding FVIII-BDD proteins are mammalian, such as human.
  • Such mammalian nucleic acids and nucleic acid variants encoding FVIII-BDD proteins include human forms, which may be based on wild-type human FVIII deleted in the B domain.
  • One aspect of the present disclosure relates to a gene expression cassette comprising, preferably consisting of, an optional enhancer, a promoter, an optional 5' untranslated region (5'UTR), an optional intron, Optional kozak sequence, B domain deleted human FVIII (FVIII-BDD) protein encoding cDNA, poly(A), and two ITRs at both ends.
  • a gene expression cassette comprising, preferably consisting of, an optional enhancer, a promoter, an optional 5' untranslated region (5'UTR), an optional intron, Optional kozak sequence, B domain deleted human FVIII (FVIII-BDD) protein encoding cDNA, poly(A), and two ITRs at both ends.
  • the gene expression cassette of the present disclosure is a gene expression cassette composed of the following elements: a promoter, a cDNA encoding a B domain-deleted human FVIII (FVIII-BDD) protein, a poly(A) A) and two ITRs located at both ends.
  • the gene expression cassette of the present invention is a gene expression cassette composed of the following elements: a promoter, a kozak sequence, a cDNA encoding a B-domain deleted human FVIII (FVIII-BDD) protein, polyadenylic acid (poly A), and two ITRs located at both ends.
  • the enhancer comprises or consists of the nucleotide sequence shown in SEQ ID NO: 8.
  • the promoter comprises or consists of the nucleotide sequence of the liver-specific promoter LXP2.1 (SEQ ID NO: 9) or LXP3.3 (SEQ ID NO: 10).
  • the 5'UTR comprises or consists of the nucleotide sequence shown in SEQ ID NO: 11.
  • the intron comprises or consists of the nucleotide sequence shown in SEQ ID NO: 12.
  • the kozak sequence contains or consists of GCCACC.
  • amino acid sequence of FVIII deleted from the B domain is, for example, SEQ ID NO: 2.
  • the B domain-deleted FVIII can be encoded by a polynucleotide comprising the nucleotide sequence shown in any one of SEQ ID NOs: 4 to 7 or having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting essentially of, or consisting of.
  • polyadenylate comprises or consists of the nucleotide sequence shown in SEQ ID NO: 13.
  • the ITR comprises or consists of the nucleotide sequence shown in SEQ ID NO: 15.
  • vectors such as expression vectors, comprising polynucleotides of the disclosure.
  • the vector may be any type of vector known in the art, including but not limited to plasmid vectors and viral vectors.
  • the viral vector is a retroviral or lentiviral vector.
  • the viral vector is an AAV vector from any known AAV serotype, including, but not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV types 7, AAV 8, AAV 9, AAV 10, AAV 11, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV and any other AAV now known or later discovered.
  • the AAV vector is AAVXL32.1 (see International Patent Publication WO 2019241324A1).
  • cells eg, isolated cells, transformed cells, recombinant cells, etc.
  • cells comprising polynucleotides and/or vectors of the disclosure.
  • some embodiments of the present disclosure relate to recombinant host cells containing vectors (eg, expression cassettes). Such cells can be isolated and/or present in transgenic animals.
  • Another aspect of the disclosure relates to transgenic animals comprising polynucleotides, vectors, and/or transformed cells of the disclosure.
  • kits comprising polynucleotides, vectors and/or cells of the present disclosure, and/or reagents and/or instructions for using the kit.
  • One aspect of the present disclosure relates to methods of treating hemophilia A or acquired factor VIII deficiency in a subject using the FVIII gene expression cassette of the present disclosure, comprising delivering to the subject a therapeutically effective amount of a polynucleotide of the present disclosure, Vectors and/or transformed cells, thereby treating hemophilia A or acquired factor VIII deficiency in a subject.
  • Another aspect of the present disclosure relates to methods for improving long-term high-efficiency expression of FVIII polypeptides in a subject using codon- and CpG-optimized nucleotide sequences of the present disclosure, comprising delivering an effective amount of a polynucleotide of the present disclosure to the subject , vectors and/or transformed cells, thereby improving long-term high-efficiency expression of FVIII polypeptides in subjects.
  • One aspect of the present disclosure relates to a method of producing a FVIII polypeptide in the liver of a subject, comprising delivering to the subject an optimized polynucleotide, a vector, and/or a transformed cell encoding a B domain deleted human FVIII polypeptide of the present disclosure, FVIII polypeptide is thereby produced in the subject's liver.
  • Another aspect of the present disclosure relates to a method of treating hemophilia A or acquired factor VIII deficiency in a subject, comprising delivering to the subject a therapeutically effective amount of an optimized polypeptide encoding a B domain deleted human FVIII polypeptide of the present disclosure.
  • the nucleotides, vectors and/or transformed cells are used to treat hemophilia A or acquired FVIII deficiency in a subject.
  • Bleeding disorders treatable according to the methods of the present disclosure include any condition treatable with FVIII, such as hemophilia A and acquired FVIII deficiency.
  • Such treatment strategies and dosing regimens for administering or delivering the FVIII proteins of the disclosure and/or polynucleotides encoding the FVIII proteins of the disclosure to a subject are within the art. Well known.
  • the dosage of the vector of the present disclosure can be an amount that achieves a therapeutic plasma concentration of the FVIII protein.
  • the therapeutic concentration of the FVIII protein is considered to be 1% higher than the normal level of a healthy individual, which is measured on an average of 100%, i.e., one international unit (IU) of FVIII in 1 mL of normal human plasma.
  • IU international unit
  • compositions of the present disclosure may be used for prophylactic and/or therapeutic administration.
  • the composition is administered to a subject already suffering from the disease as described above in an amount sufficient to cure, alleviate or partially prevent the disease and its complications.
  • An amount sufficient to achieve this goal is defined as a "therapeutically effective amount.”
  • the amount effective for this purpose will depend on the severity of the disease or injury and the weight and general condition of the subject.
  • an AAV vector is used to deliver the FVIII gene expression cassette of the present disclosure to a subject.
  • the present disclosure also provides an AAV virus particle (ie, a virion) comprising a FVIII gene expression cassette, wherein the virus particle packages a vector genome.
  • the virosome is a recombinant vector (e.g., for delivery to a cell) comprising a FVIII gene expression cassette. Therefore, the recombinant vector of the present disclosure can be used to deliver polynucleotides to cells in vitro, ex vivo, and in vivo. In representative embodiments, the recombinant vector of the present disclosure can be advantageously used to deliver or transfer the FVIII gene expression cassette of the present disclosure into animal (e.g., mammalian) cells.
  • animal e.g., mammalian
  • the present disclosure also provides methods of producing viral vectors.
  • the present disclosure provides a method of producing a recombinant viral vector, the method comprising providing to a cell in vitro (a) a template comprising (i) a polynucleotide of interest and (ii) sufficient to A packaging signal sequence (e.g., one or more (e.g., two) terminal repeats, e.g., an AAV terminal repeat) that encapsulates the AAV template into the virion; and (b) is sufficient to enable replication of the template and loading of the template into the virion.
  • AAV sequences such as AAV rep and AAV cap sequences).
  • the template and AAV replication and capsid sequences are provided under conditions such that recombinant viral particles comprising the template packaged within the capsid are produced in the cell.
  • the method may also include the step of collecting viral particles from said cells. Viral particles can be collected from the culture medium and/or by lysing cells.
  • the present disclosure provides a method of producing rAAV particles comprising an AAV capsid, the method comprising: providing to a cell in vitro a nucleic acid encoding an AAV capsid, an AAV rep coding sequence, a nucleic acid containing a target polynucleus nucleotides of the AAV vector genome, as well as helper functions for generating productive AAV infections; and allowing the assembly of AAV particles containing the AAV capsid and packaging of the AAV vector genome.
  • the cells are typically cells that are permissive for AAV viral replication. Any suitable cell known in the art may be used, such as a mammalian cell. Also suitable are trans-complementing packaging cell lines that provide functions missing from the replication-defective helper virus, such as 293 cells or other E1a trans-complementing cells.
  • AAV replication and capsid sequences can be provided by any method known in the art. Current protocols typically express the AAV rep/cap genes on a single plasmid. There is no need to provide AAV replication and packaging sequences together.
  • AAV rep and/or cap sequences can be provided by any viral or non-viral vector.
  • the rep/cap sequence may be provided by a hybrid adenoviral or herpesviral vector (eg, inserted into the E1a or E3 region of a deleted adenoviral vector).
  • EBV vectors can also be used to express AAV cap and rep genes.
  • One advantage of this approach is that the EBV vector is episomal but will maintain a high copy number throughout successive cell divisions (i.e. stably integrated into the cell as an extrachromosomal element, designated EBV-based nuclear episomal body(episome)).
  • rep/cap sequences can be stably carried within the cell.
  • AAV rep/cap sequences are not flanked by AAV packaging sequences (e.g., AAV ITR) to prevent rescue and/or packaging of these sequences.
  • AAV packaging sequences e.g., AAV ITR
  • the template (eg, rAAV vector genome) can be provided to the cell using any method known in the art.
  • the template can be provided by a non-viral (eg plasmid) or viral vector.
  • the template is provided by a herpesvirus or adenovirus vector (eg, inserted into a deleted E1a or E3 region of the adenovirus).
  • Palombo et al., (1998) J. Virol. 72:5025 describes baculovirus vectors carrying a reporter gene flanked by AAV ITRs. Templates can also be delivered using EBV vectors, as described above for the rep/cap gene.
  • the template is provided by a replicating rAAV virus.
  • the AAV provirus is stably integrated into the chromosome of the cell.
  • helper viral functions such as adenovirus or herpesvirus
  • helper viral sequences required for AAV replication are known in the art.
  • these sequences are provided by helper adenovirus or herpesvirus vectors.
  • adenoviral or herpesvirus sequences can be provided by another non-viral or viral vector, e.g. as a non-infectious adenoviral miniplasmid carrying all the helper genes required for efficient AAV production, such as Ferrari et al., ( 1997) Nature Med. 3:1295 and U.S. Patent Nos. 6,040,183 and 6,093,570.
  • helper virus function can be provided by packaging cells with helper genes integrated in the chromosome or maintained as stable extrachromosomal elements.
  • the helper viral sequences are not packaged within the AAV virion, e.g., are not flanked by the AAV ITR.
  • helper construct may be a non-viral or viral construct, but alternatively is a hybrid adenovirus or hybrid herpesvirus containing the AAV rep/cap gene.
  • the AAV rep/cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector.
  • This vector also contains rAAV template.
  • AAV rep/cap sequences and/or rAAV templates can be inserted into deleted regions of adenovirus (e.g., E1a or E3 regions).
  • the AAV rep/cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector.
  • the rAAV template is provided as a plasmid template.
  • AAV rep/cap sequences and adenoviral helper sequences are provided from a single adenoviral helper vector, and the rAAV template is integrated into the cell as a provirus.
  • the rAAV template is provided by the EBV vector maintained intracellularly as an extrachromosomal element (eg as "EBV-based nuclear episome", see Margolski, (1992) Curr. Top. Microbiol. Immun. 158:67).
  • the AAV rep/cap sequence and adenoviral helper sequence are provided by a single adenoviral helper.
  • the rAAV template is provided as a separate replicating viral vector.
  • the rAAV template can be provided by a rAAV particle or a second recombinant adenoviral particle.
  • hybrid adenovirus vectors typically contain adenovirus 5' and 3' cis-sequences (i.e., adenovirus terminal repeats and PAC sequences) sufficient for adenovirus replication and packaging.
  • AAV rep/cap sequences and (if present) rAAV template are embedded in the adenoviral backbone and flanked by the 5' and 3' cis sequences, allowing these sequences to be packaged into adenoviral capsids.
  • adenoviral helper sequences and AAV rep/cap sequences are not flanked by AAV packaging sequences (e.g., AAV ITR), such that these sequences are not packaged into AAV virions.
  • Herpes viruses can also be used as helper viruses in AAV packaging methods. Hybrid herpesviruses encoding AAV rep proteins may advantageously facilitate AAV vector production protocols. Hybrid herpes simplex virus type 1 (HSV-1) vectors expressing AAV-2 rep and cap genes have been described (Conway et al. (1999) Gene Therapy 6:986 and WO 00/17377, the disclosures of which are incorporated herein in their entirety) .
  • HSV-1 hybrid herpes simplex virus type 1
  • the viral vectors of the present disclosure can be produced in insect cells using baculovirus vectors to deliver the rep/cap gene and rAAV template, as described in Urabe et al. (2002) Human Gene Therapy 13:1935-43 narrate.
  • AAV vector stocks free of contaminating helper virus can be obtained by any method known in the art.
  • AAV and helper viruses can be easily distinguished based on their size.
  • AAV can also be isolated from helper viruses based on affinity for heparin substrate (Zolotukhin et al. (1999) Gene Therapy 6:973).
  • a deleted replication-deficient helper virus is used such that any contaminating helper virus is incapable of replication.
  • adenoviral helpers lacking late gene expression can be used, since only adenoviral early gene expression is required to mediate packaging of AAV viruses.
  • Adenovirus mutants defective in late gene expression are known in the art (eg, ts100K and ts149 adenovirus mutants).
  • the packaging methods of the present disclosure can be used to generate high titer viral particle stocks.
  • the viral stock has a titer of at least about 10 5 transduction units (tu)/ml, at least about 10 6 tu/ml, at least about 10 7 tu/ml, at least about 10 8 tu/ml, at least about 10 9 tu/ml or at least about 10 10 tu/ml.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the viral vector of the present disclosure and optional other medical agents, agents, stabilizers, buffers, carriers, adjuvants in a pharmaceutically acceptable carrier. agents, thinners, etc.
  • the carrier is usually a liquid.
  • the carrier may be solid or liquid.
  • the carrier will be respirable, and preferably will be in solid or liquid particulate form.
  • pharmaceutically acceptable refers to a substance that is not toxic or otherwise undesirable, ie, the substance can be administered to a subject without causing any undesirable biological effects.
  • Viral vectors can be introduced into cells at the appropriate multiplicity of infection according to standard transduction methods applicable to the specific target cell.
  • the titer of the viral vector or capsid used for administration can vary depending on the target cell type and number and the specific viral vector or capsid, and can be determined by one skilled in the art without undue experimentation.
  • at least about 10 3 infectious units, more preferably at least about 10 5 infectious units are introduced into the cell.
  • the cell that can introduce viral vector can be any type, including but not limited to neural cell (including peripheral and central nervous system cells, in particular, brain cells such as neurons, oligodendrocytes, glial cells, astrocytes), lung cells, eye cells (including retinal cells, retinal pigment epithelium and corneal cells), epithelial cells (such as intestinal and respiratory epithelial cells), skeletal muscle cells (including myoblasts, myotubes and myofibers), diaphragm muscle cells, dendritic cells, pancreatic cells (including islet cells), hepatocytes, gastrointestinal cells (including smooth muscle cells, epithelial cells), heart cells (including cardiomyocytes), osteocytes (such as bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, joint cells (including such as cartilage, meniscus, synovium and bone marrow), germ cells, etc.
  • neural cell
  • the cell can be any progenitor cell.
  • the cell can be a stem cell (such as a neural stem cell, a liver stem cell).
  • the cell can be a cancer or tumor cell.
  • the cell can be from a species in any source.
  • Viral vectors can be introduced into cells in vitro for the purpose of administering the modified cells to a subject.
  • cells have been removed from the subject, a viral vector has been introduced therein, and the cells have been replaced back into the subject.
  • Methods of removing cells from a subject for ex vivo treatment and then introducing them back into the subject are known in the art (see, eg, U.S. Patent No. 5,399,346).
  • the recombinant viral vector is introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need thereof. tester.
  • Suitable cells for ex vivo gene therapy are as described above.
  • the dosage of cells administered to a subject will vary depending on the age, condition and species of the subject, the type of cell, the nucleic acid expressed by the cell, the mode of administration, and the like. Typically, at least about 10 2 to about 10 8 or about 10 3 to about 10 6 cells per dose will be administered in a pharmaceutically acceptable carrier.
  • cells transduced with a viral vector are administered to a subject in an effective amount in combination with a pharmaceutical carrier.
  • Another aspect of the present disclosure relates to a method of administering a viral vector of the present disclosure to a subject.
  • the method comprises a method of delivering a target polynucleotide to an animal subject, the method comprising: administering an effective amount of a viral vector according to the present disclosure to an animal subject.
  • the viral vector of the present disclosure can be administered to a human subject or animal in need thereof by any method known in the art.
  • a viral vector in a pharmaceutically acceptable carrier is delivered at an effective dose.
  • the dose of the viral vector to be administered to a subject will depend on the mode of administration, the disease or disorder to be treated, the condition of the individual subject, the specific viral vector and the nucleic acid to be delivered, and can be determined in a routine manner.
  • Exemplary doses for achieving a therapeutic effect are at least about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 transduction units or higher viral titers, preferably about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 transduction units, and more preferably about 10 12 transduction units.
  • more than one administration may be used at variously spaced time periods (e.g., daily, weekly, monthly, yearly, etc.) Achieve desired levels of gene expression.
  • Exemplary modes of administration include oral, rectal, transmucosal, topical, intranasal, inhalation (e.g., via aerosol), intrabuccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [including administration to bone, diaphragm, and/or myocardium], intradermal, intrapleural, intracerebral, and intraarticular), topical (e.g., cutaneous and mucosal surfaces, including airway surfaces and transdermal administration), intralymphatic, etc., as well as direct tissue or organ injection (e.g., liver, skeletal muscle, myocardium, diaphragm muscle, or brain). Administration may also be given to tumors (eg, in or near tumors or lymph nodes). The most appropriate route in any given case will depend on the nature and severity of the condition being treated and
  • Delivery to any of these tissues may also be achieved by delivering a depot containing the viral vector, which may be implanted in the tissue or the tissue may be in contact with a membrane or other matrix containing the viral vector.
  • a depot containing the viral vector which may be implanted in the tissue or the tissue may be in contact with a membrane or other matrix containing the viral vector. Examples of such implantable matrices or substrates are described in US Patent No. 7,201,898.
  • the methods of the present disclosure may be used to treat disorders of tissues or organs.
  • the methods of the present disclosure can be performed to deliver nucleic acids to tissues or organs for the production of Factor VIII polypeptides that typically circulate in the blood or are delivered systemically to other tissues to treat hemophilia.
  • compositions may be presented in unit/dose or multi-dose containers, for example in sealed ampoules and vials, and may be stored under freeze-dried (lyophilized) conditions requiring only the addition of a sterile liquid carrier immediately before use, e.g. Saline or water for injection.
  • a sterile liquid carrier e.g. Saline or water for injection.
  • a vector is a replicable DNA construct.
  • Vectors are used herein to amplify a nucleic acid encoding a FVIII protein and/or to express a nucleic acid encoding a FVIII protein.
  • An expression vector is a replicable nucleic acid construct in which a nucleotide sequence encoding a FVIII protein is operably linked to a suitable control sequence that enables expression of the nucleotide sequence in a suitable host cell to produce the FVIII protein. .
  • suitable control sequence that enables expression of the nucleotide sequence in a suitable host cell to produce the FVIII protein.
  • control sequences include a transcriptional promoter, optional operator sequences for controlling transcription, sequences encoding appropriate mRNA ribosome binding sites, and sequences that control termination of transcription and translation.
  • Vectors include plasmids, viruses (eg, AAV, adenovirus, cytomegalovirus), phage, and integrable DNA fragments (i.e., fragments that are integrated into the host cell genome by recombination).
  • the vector can replicate and function independently of the host cell genome (e.g., by transient expression), or can integrate into the host cell genome itself (e.g., stable integration).
  • An expression vector may contain a promoter and RNA binding site operably linked to the nucleic acid molecule to be expressed and operable in a host cell and/or organism.
  • DNA regions or nucleotide sequences are operably linked or operably associated when they are functionally related to each other.
  • a promoter is operably linked to a coding sequence if the promoter controls the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if the promoter is positioned to allow translation of the coding sequence. this coding sequence.
  • Transcriptional and translational control sequences in expression vectors used in transforming vertebrate cells are often provided by viral sources.
  • Non-limiting examples include promoters derived from polyomavirus, adenovirus 2, and simian virus 40 (SV40). See, for example, US Patent No. 4,599,308.
  • Ubiquitous expression promoters refer to a type of strong promoters with broad activity in animal cells, tissues and cell cycles, including CMV, EF1A, EFS , CAG, CBh, CBA, SFFV, MSCV, SV40, mPGK, hPGK, UBC, etc.
  • the coding sequences of the present disclosure may encode FVIII from any species, including mouse, rat, dog, opossum, rabbit, cat, pig, horse, sheep, cow, guinea pig, opossum, platypus, and human, but preferably encode Human FVIII protein. Also included are FVIII-encoding nucleic acids that hybridize to the protein-encoding nucleic acids disclosed herein. This can be done in standard in situ hybridization assays, under reduced stringency conditions or even under stringent conditions (e.g.
  • a cis-acting regulatory region that is "active" in breast tissue may be used because the promoter is more active in breast tissue than in other tissues under the physiological conditions of synthetic milk.
  • These promoters include, but are not limited to, short and long whey acidic protein (WAP), short and long alpha, beta and kappa casein, alpha-lactalbumin and beta-lactoglobulin ("BLG”) promoters.
  • WAP short and long whey acidic protein
  • beta and kappa casein alpha-lactalbumin and beta-lactoglobulin
  • BLG beta-lactoglobulin
  • Signal sequences that secrete the expressed protein directly into other body fluids, particularly blood and urine may also be used in accordance with the present disclosure. Examples of these sequences include the signal peptides of secreted coagulation factors, including those of FVIII, protein C, and tissue plasminogen activator.
  • useful sequences for regulating transcription include enhancers, splicing signals, transcription termination signals, polyadenylation sites, buffer sequences, RNA processing sequences, and other sequences that regulate transgene expression.
  • the expression system or construct contains the 3' untranslated region downstream of the nucleotide sequence encoding the recombinant protein. This region increases transgene expression.
  • useful 3' untranslated regions are sequences that provide the poly A signal.
  • Suitable heterologous 3'-untranslated sequences may be derived from, for example, the SV40 small t antigen, the casein 3' untranslated region, or other 3' untranslated sequences well known in the art.
  • the ribosome binding site is also important for increasing the expression efficiency of FVIII.
  • sequences that modulate post-translational modifications of FVIII are useful in the present disclosure.
  • the coding sequence of the human FVIII-BDD gene FVIII-BDD-Opti was completely synthesized according to the human codon usage efficiency. Its purpose It maximizes the use of more efficient codons to produce fully synthetic human FVIII-BDD genes F8X1Co1 (SEQ ID NO: 4) and F8X1Co3 (SEQ ID NO: 6).
  • FVIII-BDD human FVIII-BDD protein in hepatocytes
  • two synthetic liver-specific promoters were selected: 188 nt LXP2.1 and 200 nt LXP3.3.
  • a synthetic transcription enhancing element with a length of only 71 nt was selected.
  • a small synthetic human intron was selected to further enhance FVIII gene expression.
  • a synthetic polyadenylate of only 48 nt was selected.
  • liver-specific promoter 5' non-coding region, intron, human FVIII-BDD encoding cDNA and polyadenylation were sequentially assembled into a gene expression cassette ( Figure 1) mediated by PEI.
  • Liver Huh7 cells were transfected in vitro, and the cell culture supernatant 3 days after transfection was used to detect human FVIII coagulation activity.
  • the gene expression cassette needs to contain a variety of regulatory elements (such as promoters, enhancers, introns, 5'UTR) to enhance gene transcription and promote gene expression.
  • regulatory elements such as promoters, enhancers, introns, 5'UTR
  • UTRs are known to play a crucial role in the post-transcriptional regulation of gene expression.
  • the inventors simplified the expression cassettes (LXP2.1-F8X1Co4 and LXP3.3-F8X1Co4) constructed in Example 1, thereby constructing a new FVIII gene expression cassette, and studied each Expression efficiency of FVIII gene expression cassette.
  • the enhancer, intron and 5'UTR in the LXP2.1-F8X1Co4 expression cassette were deleted in sequence, and the ITR was further truncated (the D sequences of the ITR at both ends were truncated by 10nt), thus Four human FVIII gene expression cassettes were obtained: LXP2.1-F8X1Co4.1, LXP2.1-F8X1Co4.2, LXP2.1-F8X1Co4.3 and LXP2.1-F8X1Co4.4 ( Figure 3).
  • the gene expression regulatory elements of LXP2.1-F8X1Co4.3 and LXP2.1-F8X1Co4.4 only retain the promoter and polyadenylic acid (poly A).
  • LXP2.1-F8X1Co4.4 in addition to removing the above elements, the D sequence of the ITR at both ends was truncated by 10 nt, and the CAGATCT sequence immediately adjacent to the D sequence was also deleted.
  • the enhancer, intron and 5’UTR in the LXP3.3-F8X1Co4 expression cassette were simultaneously deleted, thereby obtaining LXP3.3-F8X1Co4.1 ( Figure 3).
  • Each of the above expression cassettes was transfected into liver Huh7 cells in vitro through PEI mediation, and the cell culture supernatant 3 days after transfection was used to detect human FVIII coagulation activity.
  • the gene expression cassette LXP2.1-F8X1Co4.1 with the enhancer deleted did not show obvious reduction in gene expression activity, but after deleting the intron or 5'UTR, LXP2.1-F8X1Co4.2, LXP2 .1-F8X1Co4.3, LXP2.1-F8X1Co4.4 and LXP3.3-F8X1Co4.1 gene expression activities were significantly reduced, especially LXP2.1-F8X1Co4.3 and LXP2.1- whose 5'UTR was deleted.
  • F8X1Co4.4 and LXP3.3-F8X1Co4.1 their gene expression activity was reduced by even more than 80%. This result is consistent with the general expectations of those skilled in the art. It is generally believed that the removal of regulatory elements, especially the removal of the 5’ UTR that plays an important role in gene expression, will lead to a reduction in gene expression efficiency.
  • Example 3 Virus yield and genome integrity of adeno-associated virus (AAV) packaging FVIII gene expression cassette
  • human liver-tropic AAV32.1 was used to package the FVIII gene expression cassette involved in Example 2, and the virus integrity and virus yield were detected.
  • the recombinant adeno-associated viruses AAV32.1/LXP2.1-F8X1Co4, AAV32.1/LXP2.1-F8X1Co4.1, and AAV32.1/LXP2 produced by the three-plasmid suspension HEK293 system in Example 3 were used.
  • .1-F8X1Co4.2, AAV32.1/LXP2.1-F8X1Co4.3, AAV32.1/LXP2.1-F8X1Co4.4, AAV32.1/LXP3.3-F8X1Co4 and AAV32.1/LXP3.3-F8X1Co4 .1 Separate and purify through iodixanol two-step ultracentrifugation method.
  • the purified viruses were used to infect liver Huh7 cells at an MOI of 5E+5vg/cell, and the cell culture supernatant 5 days after transfection was used to detect the coagulation activity of human factor VIII.
  • AAV32.1/LXP2.1-F8X1Co4.3, AAV32.1/LXP2.1-F8X1Co4.4 and AAV32.1/LXP3.3-F8X1Co4 were found to have fewer regulatory elements in the expression cassette. 1
  • the gene expression activity produced by AAV32.1/LXP2.1-F8X1Co4, AAV32.1/LXP2.1-F8X1Co4.1, AAV32.1/LXP2.1-F8X1Co4.2 and AAV32.1 is more complete than that of gene regulatory elements.
  • /LXP3.3-F8X1Co4 is significantly higher.
  • the long-term effectiveness of the FVIII gene expression cassette LXP2.1-F8X1Co4.3 in vivo was tested.
  • Male mice with 2 to 3 months of FVIII gene knockout (hemophilia A animal model commonly used in the art) were used.
  • a standard vector dose of 2.5E+11vg/mouse was selected, and recombinant adeno-associated virus AAV32.1/LXP2.1-F8X1Co4.3 was injected into 5 model mice via the tail vein.
  • Untreated age- and sex-matched FVIII knockout mice were used as negative controls.
  • Plasma was collected via the retroorbital vein using standard methods, and plasma was collected weekly within 6 weeks after injection; plasma was collected every 4 weeks thereafter.
  • FVIII gene expression In order to quantitatively evaluate FVIII gene expression, purified recombinant human FVIII was diluted stepwise and added to the plasma of severe hemophilia A patients, as a standard curve for FVIII activity, and conventional APTT tests were performed on plasma samples.
  • the above experimental results show that the FVIII gene expression cassette of the present invention not only has a higher expression level of FVIII in vivo but also can be expressed stably and long-term in vivo.

Abstract

La présente invention concerne une séquence polynucléotidique optimisée codant pour un polypeptide du facteur VIII de coagulation humain (FVIII). La présente invention concerne également une cassette d'expression de gène FVIII, un vecteur AAV recombiné, et une composition pharmaceutique comprenant la séquence polynucléotidique optimisée, et leur utilisation dans le traitement de l'hémophilie a ou d'une déficience en facteur VIII acquise. La séquence polynucléotidique optimisée et la cassette d'expression du gène FVIII de la présente invention ont un niveau d'expression de FVIII amélioré.
PCT/CN2022/143456 2022-09-23 2022-12-29 Cassette d'expression du gène du facteur viii de coagulation humaine améliorée et son utilisation WO2024060463A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107531774A (zh) * 2015-02-06 2018-01-02 北卡罗来纳大学查珀尔希尔分校 优化的人类凝血因子viii基因表达盒及其用途
CN108884145A (zh) * 2015-11-13 2018-11-23 百深公司 用于血友病a的基因治疗的具有增加的表达的编码重组fviii变体的病毒载体
CN112538501A (zh) * 2013-09-12 2021-03-23 生物马林药物股份有限公司 腺相关病毒因子viii载体

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0911870D0 (en) * 2009-07-08 2009-08-19 Ucl Business Plc Optimised coding sequence and promoter
EP3114138B1 (fr) * 2014-03-05 2021-11-17 Pfizer Inc. Mutéines améliorées du facteur viii de coagulation
SG11201701033QA (en) * 2014-08-13 2017-03-30 Philadelphia Children Hospital An improved expression cassette for packaging and expression of variant factor viii for the treatment of hemostasis disorders
DK3198018T3 (da) * 2014-09-24 2021-03-01 Hope City Vektorvarianter af adenoassocieret virus til højeffektiv genomredigering og fremgangsmåder dertil
CN108473976B (zh) * 2015-10-28 2022-07-19 桑格摩生物治疗股份有限公司 肝特异性构建体、因子viii表达盒及其使用方法
PE20231949A1 (es) * 2015-10-30 2023-12-05 Spark Therapeutics Inc VARIANTES DEL FACTOR VIII REDUCIDO CON CpG, COMPOSICIONES Y METODOS Y USOS PARA EL TRATAMIENTO DE TRASTORNOS DE LA HEMOSTASIA
KR102625470B1 (ko) * 2017-05-31 2024-01-16 더 유니버시티 오브 노쓰 캐롤라이나 엣 채플 힐 최적화된 인간 응고 인자 ix 유전자 발현 카세트 및 이의 사용
AU2018312565A1 (en) * 2017-08-01 2020-02-27 Spark Therapeutics, Inc. Factor VIII (FVIII) gene therapy methods
AU2019325688A1 (en) * 2018-08-24 2021-03-18 Spark Therapeutics, Inc. Optimized promoter sequences, intron-free expression constructs and methods of use
US20210348135A1 (en) * 2018-10-12 2021-11-11 Genzyme Corporation Generation of improved human pah for treatment of severe pku by liver-directed gene replacement therapy
CN111808863B (zh) * 2020-06-23 2021-05-28 康霖生物科技（杭州）有限公司 一种密码子优化的凝血因子viii基因及其构建体

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112538501A (zh) * 2013-09-12 2021-03-23 生物马林药物股份有限公司 腺相关病毒因子viii载体
CN107531774A (zh) * 2015-02-06 2018-01-02 北卡罗来纳大学查珀尔希尔分校 优化的人类凝血因子viii基因表达盒及其用途
CN108884145A (zh) * 2015-11-13 2018-11-23 百深公司 用于血友病a的基因治疗的具有增加的表达的编码重组fviii变体的病毒载体

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI-JUN ZHONG, JIANG WEI, WANG FENG-SHAN, ZANG HENG-CHANG, : "Research Progress on Human Coagulation Factor Ⅷ Chinese Full Tex", FOOD AND DRUG, vol. 16, no. 3, 20 May 2014 (2014-05-20), pages 212 - 214, XP093149605 *
LIRON ELKOUBY ET AL.: "Preclinical Assessment of an Optimized AAV-FVIII Vector in Mice and Non-human Primates for the Treatment of Hemophilia A", MOLECULAR THERAPY - METHODS & CLINICAL DEVELOPMENT, vol. 24, 10 March 2022 (2022-03-10), pages 20 - 29, XP093069094, DOI: 10.1016/j.omtm.2021.11.005 *

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